USPTO Patents Application 10687850

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization

International Bureau

(43) International Publication Date
4 October 2001 (04.10.2001)

PCT

(10) International Publication Number

WO 01/73114 A2

(51) International Patent Classification 7 : C12Q l/32 t 1/26

(21) International Application Number: PCT/US0 1/076 19

(22) International Filing Date: 8 March 2001 (08.03.2001 )

(25) Filing Language: English

(26) Publication Language: English

(30) Priority Data:

09/537,203

28 March 2000 (28.03.2000) US

(71) Applicant: LIFESCAN, INC. [US/US]; 1000 Gibraltar
Drive, Milpitas, CA 95035-6312 (US).

(72) Inventor: OUYANG, Tianmei; 41945 Via San Gabriel,
Fremont, CA 94539 (US).

(74) Agent: FIELD, Bret, E.; Bozicevic, Field & Francis LLP,
Suite 200, 200 Middlefield Road, Menlo Park, CA 94025
(US).

(81) Designated States (national): AE, AG. AL, AM, AT, AU,
AZ, BA, BB, BC BR, BY, BZ, CA, CH, CN, CR. CU, CZ,
DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR,
HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
LS, LT, LU, LV, MA, MD, MG, MK. MN, MW, MX, MZ,
NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK. SL, TJ, TM,
TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW.

(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
, IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI. CM, GA, GN, GW, ML, MR, NE, SN, TD. TG).

Published:

— without international search report and to be republished
upon receipt of that report

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.

(54) Title: REAGENT SYSTEMS FOR DETECTING THE PRESENCE OF A REDUCED COFACTOR IN A SAMPLE AND
METHODS FOR USING THE SAME

(K/S

Mr sec;

' = B. 4406

f

t* 0.0335

I

/

at ft 13 0l2 (L29 QJ

Theoretical Rata; Tne predicted rate
dtephorase and PUS individually.

Conclusion: The reaction rata is accelerated

based on the mm of the r ea ctio n rats catalyzed by
and PMS together.

by iiuxiJiQ

£5 (57) Abstract: Signal producing systems, reagent compositions, test strips and kits of the same, as well as methods for their use in
— the detection of an analyte in a sample, are provided. The subject signal producing systems are characterized by having at least a first
^ and second electron transfer agent and a redox indicator, where in many preferred embodiments the systems include a proteinaceous
and non-proteinaceous electron transfer agent, e.g. a phenazine compound and a diaphorase. In many preferred embodiments, the
subject systems and kits further include at least one of and often both of an enzyme cofactor and an enzyme having an analyte
oxidizing activity, e.g. an analyte dehydrogenase. The subject systems, reagent compositions, test strips and kits find use in the
^ detection of a wide variety of analytcs in a sample, such as a physiological sample, e.g. blood or a fraction thereof.

WO 01/731 14

PCT/US01/07619

REAGENT SYSTEMS FOR DETECTING THE PRESENCE OF A REDUCED
COFACTOR JN A SAMPLE AND METHODS FOR USING THE SAME

INTRODUCTION!

Field of the Invention

The field of this invention is analyte detection, particularly reagent systems for use in
analyte detection.

Background of the Invention

Analyte detection in physiological fluids, e.g. blood or blood derived products, is of
ever increasing importance to today's society. Analyte detection assays find use in a variety
of applications, including clinical laboratory testing, home testing, etc, where the results of
such testing play a prominent role in diagnosis and management in a variety of disease
conditions. Analytes of interest include alcohol, formaldehyde, glucose, glutamic acid,
glycerol, beta-hydroxybutyrate, L-lactate, leucine, malic acid, pyruvic acid, steroids, etc. In
response to this growing importance of analyte detection, a variety of analyte detection
protocols and devices for both clinical and home use have been developed. Many of the
protocols and devices that have been developed to date employ a signal producing system to
identify the presence of the analyte of interest in a physiological sample, such as blood.

One type of signal producing system that finds use in the detection of a variety of
different analytes is one in which a dehydrogenase oxidizes the analyte of interest and
concomitantly reduces an enzyme cofactor, such as NAD(P)+. The reduced form of the
cofactor, e.g. NAD(P)H, is then detected through subsequent reaction with a cofactor
oxidizing agent, e.g. phenazine methosulfate or a diaphorase, that transfers an electron to a
redox indicator, such as a tetrazolium salt, to produce a detectable product.

While a variety of such signal producing systems have been developed to date for use
in the detection of a wide variety of different analytes, there continues to be a need for the
further development of such systems. For example, a signal producing system which
provided for an enhanced reaction rate and a lower cost would be of great interest.
Relevant Literature

U.S. Patents of interest include: 4,629,697; 5,126,247 and 5,902,731. See also Raap
et al., Histochem. J. (1983) 15:881-893.

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SUMMARY OF THE INVENTION
Signal producing systems, reagent compositions, test strips and kits of the same, as
well as methods for their use in the detection of an analyte in a sample, are provided. The
subject signal producing systems are characterized by having at least a first and second

5 electron transfer agent and a redox indicator, where in many preferred embodiments the
systems include a proteinaceous and non-proteinaceous electron transfer agent, e.g. a
phenazine compound and a diaphorase. In many preferred embodiments, the subject systems
and kits further include both an enzyme cofactor and an enzyme having an analyte oxidizing
activity, e.g. an analyte dehydrogenase. The subject systems, reagent compositions, test

10 strips and kits find use in the detection of a wide variety of analytes in a sample, such as a
physiological sample, e.g. blood or a fraction thereof.

BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 provides a graphical representation of the observed rate of reaction in a test
15 strip according to the subject invention vs. the theoretical expected rate of reaction for a test
strip that includes both PMS and a diaphorase, clearly demonstrating that the use of both a
non-proteinaceous and proteinaceous electron transfer agent, e.g. PMS and a diaphorase,
provides for an unexpected increase in the rate of reaction.

20 DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Signal producing systems, reagent compositions, test strips and kits of the same, as
well as methods for their use in the detection of an analyte in a sample, are provided. The
subject signal producing systems are characterized by having at least a first and second
electron transfer agent and a redox indicator, where in many preferred embodiments the

25 systems include a proteinaceous and non-proteinaceous electron transfer agent, e.g. a

phenazine compound and a diaphorase. In many preferred embodiments, the subject systems
and kits further include both an enzyme cofactor and an enzyme having an analyte oxidizing
activity, e.g. an analyte dehydrogenase. The subject systems, reagent compositions, test
strips and kits find use in the detection of a wide variety of analytes in a sample, such as a

30 physiological sample, e.g. blood or a fraction thereof

Before the subject invention is described further, it is to be understood that the
invention is not limited to the particular embodiments of the invention described below, as

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10

15

variations of the particular embodiments may be made and still fall within the scope of the
appended claims. It is also to be understood that the terminology employed is for the purpose
of describing particular embodiments, and is not intended to be limiting. Instead, the scope
of the present invention will be established by the appended claims.

In this specification and the appended claims, singular references include the plural,
unless the context clearly dictates otherwise. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs.

Signal Producing Systems

As summarized above, the subject invention provides a signal producing system that
is capable of detecting the presence of a reduced enzyme cefaclor in a sample. By signal
producing system is meant a collection of two or more compounds or molecules which are
capable of acting in concert, when combined, to produce a detectable signal that is indicative
of the presence of, and often amount of, a particular analyte in a given sample. The term
signal producing system is used broadly to encompass both a mixture of all of the reagent
constituents of the signal producing system as well as a system in which one or more of the
reagent constituents are separated from the remainder of the reagent constituents, e.g. as is
20 present in a kit.

A feature of the subject signal producing systems is the presence of two distinct
electron transfer agents. By electron transfer agent is meant a compound or molecule that
can transfer an electron, in the form of a hydride ion, from a reduced enzyme cofactor to a
redox indicator. In the subject signal producing systems, the first of the distinct electron

25 transfer agents is a low molecular weight molecule, while the second electron transfer agent
is a high molecular weight molecule. In this specification, low molecular weight means a
molecular weight that does not exceed about 2000 daltons, usually about 1000 daitons and in
many embodiments about 500 daltons. High molecular weight means a molecular weight of
at least about 5000 daltons and in many embodiments 10,000 or 20,000 daltons or higher.

30 The molecular weight of the high molecular weight electron transfer agent often will not
exceed about 100,000 daltons. In many embodiments, the low molecular weight electron
transfer agent is a non-proteinaceous compound while the high molecular weight electron

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transfer agent is a proteinaceous compound. By proteinaceous is meant a polypeptide or
polymeric mimetic thereof.

A variety of low molecular weight non-proteinaceous electron transfer agents are of
interest. These agents include: flavins such as riboflavin (RBF), alloxazine

5 (ALL) and lumichrome (LC); phenazines such as phenazine, phenazine methosulfate (PMS),
phenazine ethosulfate, methoxyphenazine methosulfate and safranine; methyl- 1, 4-naphthol
(menadione), phenothiazines such as PT and its radical cation, PT+, thionin (TH), azure A
(AA), azure B (AB), azure C (AC), methylene blue (MB), methylene green (MG) and
toluidine blue 0 (TOL); phenoxazines such as phenoxazine (PQA), basic blue 3 (BB3), and

10 brilliant cresyl blue ALD (BCBA), benzo-ct-phenazoxonium chloride (Medola's blue);
Indophenols such as 2,6-dichlorophenol indophenol (DCEP); and Indamines such as
Bindschedler's green and phenylene blue; and the like.: Of particular interest in many
embodiments are phenazine compounds, e.g. PMS, phenazine ethosulfate,
methoxyphenazine methosulfate and safranine, where PMS is the low molecular weight,

15 non-proteinaceous electron transfer agent in many embodiments.

In many embodiments, the high molecular weight proteinaceous electron transfer
agent is an enzyme that is capable of oxidizing a reduced cofactor, e.g. NAD(P)H, and
concomitantly reducing a redox indicator. In many embodiments, this electron transfer
enzyme is a diaphorase, such as lipoic dehydrogenase, ferredoxin-NADP reductase,

20 lipoamide dehydrogenase, N ADPH dehydrogenase, etc. A variety of diaphorases are

available and may be employed, where representative commercially available diaphorases
that may be present in the subject signal producing systems include bacillus diaphorase,
Clostridium diaphorase, vibrio diaphorase, porcine diaphorase, and the like.

In the subject signal producing systems, the ratio of the first to the second electron

25 transfer agent is chosen to provide for an accelerated reaction rate as compared to a control,
e.g. a comparable signal producing system with a single electron transfer agent, e.g. only
PMS or a diaphorase. Typically, the ratio of the first to the second electron transfer agent in
the subject systems ranges from about 0.001 to 10, usually from about 0.01 to 1.0 and more
usually from about 0.05 to 0.5 (nmole/U), respectively.

30 In addition to the above described first and second electron transfer agents, the

subject signal producing systems also include a redox indicator. By redox indicator is meant
a compound that is capable of being reduced by the electron transfer agents to produce a
detectable, e.g. chromogenic, product. Where the redox indicator produces a chromogenic

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product, i.e. the redox indicator is a chromogen, suitable chromogens are any compound
capable of changing color upon reduction by one or more electrons, where suitable
chromogens are generally ones that accept electrons from the electron transfer agents,
described above.

A variety of different redox indicator compounds are of interest. Compounds of
interest include: oxazines, thiazines, and tetrazolium salts. Of particular interest in many
embodiments are tetrazolium salts which are capable of accepting the captured hydride from
the electron transfer agents to form a colored formazan product. In many embodiments, these
salts have the advantageous feature of being faint yellow in the oxidized form, but turn
bright visible colors upon electron reduction and conversion to formazan dyes. Tetrazolium
compounds or salts that are of particular interest include: 2-(2' benzothiazolyl)-5-styryl-3-
(4'-phthalhydrazidyl) tetrazolium (BSPT); 2-benzothiazolyl-(2)-3,5-diphenyl tetrazolium
(BTDP), 2,3-di(4-nitrophenyl) tetrazolium (DNP); 2,5-diphenyl-3-(4-styrylphenyl)
tetrazolium (DPSP); distyryl nitroblue tetrazolium (DS-NBT); 3,3'-[3,3'-dimethoxy-(l,l'-
biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5- phenyI(-2H tetrazolium (NBT); 3-(4,5-
dimethyI-2-thiazolyl)-2,5-diphenyl-2H tetrazolium (MTT); 2-phenyl-3-(4-carboxyphenyl)-5-
methyl tetrazolium (PCPM); tetrazolium blue (TB); thiocarbamyl nitroblue tetrazolium
(TCNBT); tetranitroblue tetrazolium (TNBT); tetrazolium violet (TV); 2-

benzothiazothiazolyl-3-(4-carboxy-2-methoxyphenyI)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-
2H-tetrazolium (WST-4); 2,2'-dibenzothiazolyI-5,5'-bis[4-di(2-

sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylene)ditetrazolium,
disodium salt (WST-5); 2-(p-nitrophenyl)-3-(p-iodophenyl)-5-phenyltetrazolium chloride
(INT); and the like. WST-5 is preferred in many embodiments because it readily dissolves in
an aqueous medium, which is most compatible with biological samples. Furthermore, the
resulting formazan compound exhibits strong spectral absorption at the purple-blue region,
thus reducing the need for correcting the background signal from hemoglobin. Other useful
tetrazolium salts are disclosed in U.S. Pat. Nos. 4,490,465; 4,491,63 1; 4,598,042; 4,351,899;
4,271,265; 4,247,633; 4,223,090; 4,215,917; 4,142,938; 4,024,021; 3,867,259; 3,867,257;
3,791,931; and 4,254,222; the disclosures of which are herein incorporated by reference.

The above described signal producing systems are capable of detecting the presence
of a reduced enzyme cofactor in a sample, particularly an aqueous sample and more
particularly a physiological sample, e.g. whole blood or a fraction or derivative thereof. A
variety of different reduced enzyme cofactors may be detected using the subject signal

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producing systems, where representative reduced enzyme cofactors include the reduced
forms of the following cofactors: beta-nicotinamide adenine dinucleotide (beta-NAD), beta-
nicotinamide adenine dinucleotide phosphate (beta-NADP), thionicotinamide adenine
dinucleotide, thionicotinamide adenine dinucleotide phosphate, nicotinamide 1,N6-
5 ethenoadenine dinucleotide, nicotinamide l,N6-ethenoadenine dinucleotide phosphate, and
pyrrolo-quinoline quinone (PQQ). The subject signal producing systems are particularly
suited for use in the detection of NADH or NAD(P)H.

In many applications in which the subject signal producing systems find use, the
reduced enzyme cofactor is one that is produced following the oxidation of an analyte of

10 interest in a sample. In many embodiments, therefore, the subject signal producing systems
also include the enzyme cofactor and an analyte oxidizing enzyme that is capable of
oxidizing the analyte of interest and concomitantly reducing the enzyme cofactor. Enzyme
cofactors of interest include those described above, i.e. beta-nicotinamide adenine
dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate (beta-NADP),

15 thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide phosphate,
nicotinamide l,N6-ethenoadenine dinucleotide, nicotinamide 1 ,N6-ethenoadenine
dinucleotide phosphate, and pyrrolo-quinoline quinone (PQQ). Enzyme cofactors of
particular interest that may be included in the subject signal producing systems include:
NADH or NAD(P)H.

20 The analyte oxidizing enzyme present in the signal producing system necessarily

depends on the nature of the analyte to be detected with the system. Representative analyte
oxidizing enzymes of interest include: alcohol dehydrogenase for alcohol, formaldehyde
dehydrogenase for formaldehyde, glucose dehydrogenase for glucose, glucose-6-phosphate
dehydrogenase for glucose-6-phosphate, glutamate dehydrogenase for glutamic acid,

25 glycerol dehydrogenase for glycerol, beta-hydroxybutyrate dehydrogenase for beta-

hydroxybutyrate, hydroxysteroid dehydrogenase for steroid, L-lactate dehydrogenase for L-
lactate, leucine dehydrogenase for leucine, malate dehydrogenase for malic acid, and
pyruvate dehydrogenase for pyruvic acid. As can be seen from the above representative list,
the analyte oxidizing enzyme is typically a dehydrogenase.

30

Reagent Compositions

Also provided by the subject invention are reagent compositions for use in detecting
at least a reduced enzyme cofactor, and in many embodiments and analyte, in a sample. The

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reagent compositions may be fluid, e.g. aqueous, or dry compositions, where in many
embodiments the reagent compositions are dry compositions. At a minimum, the subject
reagent compositions are ones that include the first and second electron transfer agent and
the redox indicator, where these components are described above. Such reagent
compositions are suitable for use in the detection of reduced enzyme cofactors, e.g.
NAD(P)H, in a sample. In many embodiments, however, the reagent compositions further
mclude an enzyme cofactor and an analyte oxidizing enzyme, where these components are
described above.

Reagent Test Strips

Also provided by the subject invention are reagent test strips for use in detecting the
presence of an analyte in a sample. In particular, the invention provides dry strips for
assaying for a particular analyte in whole blood, e.g. in beta-hydroxybutyrate, glucose, etc.
In the broadest sense, the reagent test strip includes a solid support and a dry reagent
composition present thereon, where the dry reagent composition is made up of all of the
reagent compounds necessary to produce a detectable signal in the presence of the analyte of
interest. In most embodiments of the subject invention, the dry reagent composition present
on the subject test strip is one that includes the following members: an analyte oxidizing
enzyme, an enzyme cofactor, first and second electron transfer agents and a redox indicator,
where each of these constituent members are described in greater detail supra.

In many embodiments, the subject test strips include a membrane test pad that is
affixed to a solid support. The support may be a plastic - e.g., polystyrene, nylon, or
polyester - or metallic sheet or any other suitable material known in the art. In many
embodiments, the test pad preferably comprises a bibulous, such as filter paper or polymer
membrane. Associated with the test pad, e.g. coated onto the test pad, incorporated into the
test pad, etc., is the reagent composition. The strip may also be configured in more complex
arrangements, e.g. where the test pad is present between the support and a surface layer,
where one or more reagents employed in sample processing may be present on the surface
layer. In addition, flow paths or channels may be present on the test strip, as is known in the
art. Of interest in many embodiments are the test strip configurations disclosed in U.S.
Patent No. 5,902,731, the disclosure of which is herein incorporated by reference.

The subject test strips may be fabricated employing any convenient protocol. One
convenient protocol is to contact at least the test pad portion of the strip with an aqueous

WO 01/73114 PCT/US01/07619

composition that includes all of the members of the reagent composition that is to be
associated with the test pad in the final reagent test strip. Conveniently, the test pad may be
immersed in the aqueous composition, maintained therein for a sufficient period of time and
then dried, whereby the test pad of the reagent test strip which has associated therewith the

5 reagent composition is produced. As stated above, the aqueous composition will include the
various members of the reagent composition to be associated with the test pad of the reagent
test strip, where the various members are present in amounts sufficient to provide for the
desired amounts in the reagent composition that is produced on the test pad. As such, the
concentration of non-proteinaceous electron transfer agent present in this aqueous

10 composition typically ranges from about 1 jiM to 1000 pM, usually from about 10 jiM to
500 liM; while the concentration of proteinaceous electron transfer agent in the aqueous
composition ranges from about SOU to 3000 U, usually from about 100 U to 1000 U. The
concentration of redox reagent present in the aqueous composition ranges from about 3 mM
to 36 mM, usually from about 6 mM to 24mM. When present, the enzyme cofactor ranges in

15 concentration from about 1.5 mM to 28 mM, usually from about 3.5mM to 14 mM.

Similarly, the analyte oxidizing agent enzyme ranges in concentration from about 100 U to
2000 U, and usually from about 200 U to 1000 U when present. Other components that may
be present in this aqueous composition employed to prepare the reagent test strip include
sodium chloride, magnesium chloride, Tris, PSSA, Tetronic 1307, Crotein-SPA, sucrose,

20 oxamic acid, sodium salt, and the like. See the experimental section, infra, for a more

detailed description of a representative method for preparing the subject reagent test strips.

Methods of Analyte detection

The above described signal producing systems, reagent compositions and test strips

25 find use in methods of detecting the presence of, and often the amount of, an analyte in a
sample. A variety of different analytes may be detected using the subject methods, where
representative analytes include those described above, e.g. alcohol, formaldehyde, glucose,
glutamic acid, glycerol, beta-hydroxybutyrate, L-iactate, leucine, malic acid, pyruvic acid,
steroids, etc. While in principle, the subject methods may be used to determine the presence,

30 and often concentration, of an analyte in a variety of different physiological samples, such as
urine, tears, saliva, and the like, they are particularly suited for use in determining the
concentration of an analyte in blood or blood fractions, e.g. blood derived samples, and more
particularly in whole blood.

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An important feature of the subject methods is that use of the subject signal
producing systems that include both first and second electron transfer agents provides for an
accelerated reaction time as compared to a control system, e.g. a system that includes a
single electron transfer agent. Generally, the reaction rate is accelerated or enhanced by a
5 factor of 1 to 3 depending on the ratio of PMS and diaphorase. Furthermore, the reaction rate
is greater than the theoretical or predicted rate which would be expected based on the
summation of the rates provided by the individual electron transfer agents by a factor of I
to3. Where the reaction rate is measured in terms of K/S per sec (see the experimental
section infra) the K/S per sec for a reaction in which the subject signal producing systems
10 are employed typically ranges from about 0.01 to 10, usually from about 0.05 to 5 and more
usually from about 0. 1 to 2.

In the subject methods, the sample and the signal producing system are combined
into a reaction mixture, the reaction is allowed to proceed for a sufficient period to time to
generate a signal indicative of the presence of (and often amount of) analyte in the sample,

15 and the resultant signal is detected and related to the presence of (and often amount of)
analyte in the sample. In the broadest sense, the reaction mixture may be produced in any
convenient environment, such as a cuvette or other fluid containment means. However, in
many embodiments, the above steps take place on a reagent test strip as described supra. As
such, the subject methods are now discussed further in terms of methods in which a reagent

20 test strip is employed.

In practicing the subject methods, the first step is to apply a quantity of the
physiological sample to the test strip, where the test strip is described supra. The amount of
physiological sample, e.g. blood, that is applied to the test strip may vary, but generally
ranges from about 2[iL to 40uL, usually from about 5uL to 20uL. Because of the nature

25 of the subject test strip, the blood sample size that is applied to the test strip may be

relatively small, ranging in size from about 2uL to 40jiL, usually from about 5uL to 20uL.
Where blood is the physiological sample, blood samples of a variety of different hematocrits
may be assayed with the subject methods, where the hematocrit may range from about 20%
to 65%, usually from about 25% to 60%.

30 Following application of the sample to the test strip, the sample is allowed to react

with the members of the signal producing system to produce a detectable product that is
present in an amount proportional to the initial amount of the analyte of interest present in
the sample. The amount of detectable product, i.e. signal produced by the signal producing

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system, is then determined and related to the amount of analyte in the initial sample. In
certain embodiments, automated instruments that perform the above mentioned detection
and relation steps are employed. The above described reaction, detection and relating steps,
as well as instruments for performing the same, are further described in U.S. Patent Nos.

5 4,734,360; 4,900,666; 4,935,346; 5,059,394; 5,304,468; 5,306,623; 5,418,142; 5,426,032;
5,515,170; 5,526,120; 5,563,042; 5,620,863; 5,753,429; 5,573,452; 5,780,304; 5,789,255;
5,843,691; 5,846,486; 5,902,731; 5,968,836 and 5,972,294; the disclosures of which are
herein incorporated by reference. In the relation step, the derived analyte concentration
takes into account the constant contribution of competing reactions to the observed signal,

10 e.g. by calibrating the instrument accordingly.

Kits

AJso provided by the subject invention are kits for use in practicing the subject
methods. The kits of the subject invention at least include a signal producing system as

15 described above, where the signal producing system components may be combined into a
single reagent composition or separated, e.g. present in separate containers. In certain
embodiments, the signal producing system will be present in the kits in the form of a reagent
test strip, as described supra. The subject kits may further include a means for obtaining a
physiological sample. For example, where the physiological sample is blood, the subject kits

20 may further include a means for obtaining a blood sample, such as a lance for sticking a
finger, a lance actuation means, and the like. In addition, the subject kits may include a
control solution or standard, e.g. an analyte control solution that contains a standardized
concentration of analyte. In certain embodiments, the kits also include an automated
instrument, as described above, for detecting the amount of product produced on the strip

25 following sample application and relating the detected product to the amount of analyte in
the sample. Finally, the kits include instructions for using the subject kit components in the
determination of an analyte concentration in a physiological sample. These instructions may
be present on one or more of the packaging, a label insert, containers present in the kits, and
the like.

30

The following examples are offered by way of illustration and not by way of
limitation.

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EXPERIMENT AT
A. Preparation of Ketone Test Strip

An 0.8 \xm nylon membrane obtained from Cuno (Meridien, CT) was dipped into the
reagent of Table 1, until saturated.

Table 1

Component

Quantity

100 ml

Tris(hydroxymethyl)aminomethane

sodium chloride (MW 56.44, Sigma St. Louis. MO)

1.2 gm

Magnesium Chloride (MW 203, Sigma, St. Louis, MO)

560 mg

PSSA, polystyrenesulfonic acid, sodium salt (MW 70.000)

Crotein-SPA (Croda Inc M Parsippany, NJ)

Oxamic acid, sodium salt

Tetronic 1307 (BASF Corporation, Mount Olive, NJ)

1 » wi*rw. l^J/

Sucrose (MW 342.30, Aldrich Chemicals. M ilwaukee WI)

leg

3 gm

250 mg

2 gm

NAD (MW 663.4, N7004. Sipma. St. Louis. MO)

5 gm

D-3-hydroxybutyrate dehydrogenase

450 mg

50.000 U

WST-5 (MW 1331.37. Dojindo, Japan)

Diaphorase

1.8 gm

Phenazine Methosulfate (PMS)

0-15000 U

0-3 mg

The excess reagent was scraped off gently with a glass rod. The resulting membrane was
hung to dry in a 56 °C oven for 10 minutes. Porex (0.6 mm thick) was soaked in the 5%
nitrite solution and then hung to dry in a 100 °C oven for ten hours. Finally, the membrane
was laminated between a polyester stock (0.4 mm Melenex® polyester from ICI America,
Wilmington DE) and the nitrite impregnated Porex.

B. Assays

Using the following protocol, 10 of aqueous samples comprising 40mg/dL p) 0-
hydroxybutyrate were tested on strips as described above, where the strips varied in terms of
the amount of PMS and/or Diaphorase present on the strip. A 10 ml aqueous sample was
applied onto a freshly prepared test strip. The strip was inserted into a reflectometer and
data acquisition was commenced. The relectance of the reading strip was monitored at 660
nm at one-second intervals for two minutes. Next, the data were uploaded from the ,
reflectometer's memory buffer to a personal computer via a modified serial cable. The
reaction rate was calculated based on the initial rate of change in K/S at the range where the
reaction profile was linear. The results summarized in Table 2 were averages of five
replicates.. In each individual assay, the reaction rate (in terms of K/S per sec) was observed.

n

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(K/S is a measure of reflectance, discussed and defined in USP 4,935,346, col. 14, the
disclosure of which is herein incorporated by reference.) Table 2 provides the results.

Table 2

DAD and PMS Formulation Separately

DAD and PMS Formulated

Together

DAD

Rate

PMS

Rate

Separate!

DAD

PMS

Together

U/ml

(K/S/sec)

(nm)

(K/S/sec)

(K/S/sec)

U/ml

(urn)

(K/S/sec)

150

0.0166

10

0.013

0.0296

150

10

0.0385

300

0.0392

20

0.0225

0.0617

300

20

0.1054

600

0.0943

40

0.0484

0.1427

600

40

0.3095

900

0.1666

60

0.078

0.2446

900

60

0.5861

1200

0.1787

80

0.1085

0.2872

1200

80

0.9107

1500

0.2866

100

0.13

0.4166

1500

100

Too fast*

t Theoretical rate, i.e. the predicted rate based on the sum of the reaction rate catalyzed by

5 diaphorase and PMS individually.

* The reaction is too fast and difficult to calculate the rate precisely.

C. Observed Rate v. Theoretical Rate

Table 3 below provides a comparison of the observed rate and the expected or
10 theoretical rate for the above described assays.

Table 3

Theoretical Rate (K/S per sec)

Observed Rate (K/S per sec)

0.0296

0.0385

0.0617

0.1054

0.1427

0.3095

0.2446

0.5861

0.2872

0.9107

Fig. 1 provides a graph of the observed rate vs. the theoretical rate.
As can be seen by the graph of Figure 1, the reaction rate of the signal producing
15 system of the test strip is accelerated by the presence of both PMS and Diaphorase, where
the magnitude of the observed acceleration is unexpectedly greater than the predicted
amount of acceleration based on the sum of the reaction rates of systems having PMS or
Diaphorase individually.

20 It is evident from the above results and discussion that the subject invention provides

for a significant and unexpected enhancement in the rate of reaction observed in an analyte
detection protocol based on the oxidation of an analyte and the concomitant reduction of a
redox indicator. In addition, the subject invention provides for a more economical manner of
analyte detection, as compared to certain prior art methods, e.g. ones that rely solely on

12

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diaphorase as the electron transfer agent. As such, the subject invention represents a
significant contribution to the art.

All publications and patents cited in this specification are herein incorporated by
reference as if each individual publication or patent were specifically and individually
indicated to be incorporated by reference. The citation of any publication is for its disclosure
prior to the filing date and should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior invention.

Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it is readily apparent to
those of ordinary skill in the art in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the spirit or scope of the
appended claims.

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WHAT IS CLAIMED IS:

1 . A signal producing system for detecting the presence of a reduced cofactor in a
sample, said system comprising;

5 first and second electron transfer agents capable of oxidizing a reduced cofactor; and

a redox indicator.

2. The signal producing system according to Claim 1, wherein said first electron
transfer agent is a low molecular weight electron transfer agent.

10

3. The signal producing system according to Claim 2, wherein said low molecular
weight electron transfer agent is a phenazine compound.

4. The signal producing system according to Claim 1, wherein said second electron
15 transfer agent is a high molecular weight electron transfer agent.

5. The signal producing system according to Claim 1, wherein said high molecular
weight electron transfer agent is a proteinaceous compound.

20 6. The signal producing system according to Claim 5, wherein said proteinaceous
compound is an enzyme.

7. The signal producing system according to Claim 1, wherein said redox indicator is a
tetrazolium compound.

25

8. The signal producing system according to Claim 1, wherein said signal producing
system further comprises an enzyme cofactor.

9. The signal producing system according to Claim 1, wherein said system further
30 comprises an analyte oxidizing enzyme.

10. The signal producing system according to Claim 1, wherein said signal producing
system is present as a reagent composition.

14

WO 01/73,14 PCT/US01/076I9

1 1. A reagent composition for use in detecting the presence of an analyte in a sample,
said composition comprising:

an analyte oxidizing enzyme;

an enzyme cofactor;

a non-proteinaceous electron transfer agent;
a proteinaceous electron transfer agent; and
a redox indicator.

12. The reagent composition according to Claim 11, wherein said enzyme cofactor is
NAD(P)+

13. The reagent composition according to Claim 1 1, wherein said non-proteinaceous
electron transfer agent is a phenazine compound.

14. The reagent composition according to Claim 1 1, wherein said proteinaceous electron
transfer agent is a diaphorase.

15. The reagent composition according to Claim 11, wherein said redox indicator is a
tetrazolium compound.

16. The reagent composition according to Claim 1 1, wherein said composition is a dry
reagent composition.

17. A reagent composition for use in detecting the presence of an analyte in a sample,
said composition comprising:

an analyte dehydrogenase
NAD(P)+;

a phenazine compound;

a diaphorase; and

a tetrazolium compound.

18. The reagent composition according to Claim 1 7, wherein said phenazine compound
is phenazine methosulfate.

15

WO 01/73114 PCT/US01/07619

19. The reagent composition according to Claim 17, wherein said composition is a dry
reagent composition.

20. The reagent composition according to Claim 19, wherein said dry reagent
5 composition is present on a test strip.

21 . A reagent test strip comprising:

(a) a support element; and

(b) a dry reagent composition comprising:

10 (i) an analyte oxidizing enzyme;

(ii) an enzyme cofactor;

(iii) a non-proteinaceous electron transfer agent;

(iv) a proteinaceous electron transfer agent; and

(v) a redox indicator.

15

22. The reagent test strip according to Claim 21, wherein said first enzyme is a
dehydrogenase.

23. The reagent test strip according to Claim 21, wherein said cofactor is NAD(P)+.

20

24. The reagent test strip according to Claim 21, wherein said non-proteinaceous electron
transfer agent is a phenazine compound.

25. The reagent test strip according to Claim 24, wherein said phenazine compound is
25 phenazine methosulfate.

26. The reagent test strip according to Claim 21 , wherein said proteinaceous electron
transfer agent is a diaphorase.

30 27. The reagent test strip according to Claim 21, wherein said redox indicator is a
tetrazolium compound.

16

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28. In a method for detecting the presence of an analyte in a sample, the improvement
comprising:

employing a signal producing system according to Claim

5 29. The method according to Claim 28, wherein said signal producing system is present
as a reagent composition according to Claim 1 1

30, The method according to Claim 29, wherein said reagent composition is present on a
reagent test strip according to Claim 21.

31. A kit for use in detecting the presence of an analyte in a sample, said kit comprising:
a signal producing system according to Claim 1.

32. The kit according to Claim 31, wherein said signal producing system is present as a
reagent composition according to Claim 11.

33. The kit according to Claim 32, wherein said reagent composition is present on a
reagent test strip according to Claim 21 .

34. The kit according to Claim 3 1 , wherein said kit further comprises a means for
obtaining a physiological sample.

35. The kit according to Claim 34, wherein said means for obtaining a physiological
sample is a lance;

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1/1
Figure 1

1

0.9

o.a

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

Observed Rate (K/S per sec) vs Theoretical Rate

(K/S

Der sec]

*

i

'=8.4406

x 2 * 0.483
t 2 « 0.963

< + 0.0335

F

)

f ♦

—

0.05 0.1 0.15 0.2 0.25

Theoretical Rate (K/S per sec)

0.3

0.35

Theoretical Rate: The predicted rate based on the sum of the reaction rate catalyzed by
diaphorase and PMS individually.

Observed Rate: The reaction rate catalyzed by mixing diaphorase and PMS together.
Conclusion: The reaction rate is accelerated by mixing diaphorase and PMS together.

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